Project Summary/Abstract: X-linked hypophosphatemia (XLH) is the most common hereditary hypophosphatemic disorder resulting from loss-of-function of the phosphate-regulating endopeptidase, PHEX. In the craniofacial region the most notable defect is the rachitic tooth, which presents with weak hypomineralized dentin, brittle enamel, and defective cementum. These defects ultimately lead to attachment loss and spontaneous abscesses that result in premature edentulism. Although the mineralization defects observed in XLH patients have been solely attributed to decreased serum phosphate levels, the local disruption in the organization of the dentin matrix that precedes and sustains mineralization has not been studied. Neverthesless, disruption of extracellular matrix (ECM) factors necessary for the maturation and mineralization of cartilage, bone and cementum have been reported in Hyp mice, the animal model for XLH, suggesting that PHEX mutations and/or hypophosphatemia may indeed disrupt ECM deposition. An increasing amount of evidence suggests that PHEX and the non-collagenous proteins of bone and teeth converge on a common, albeit poorly defined, pathway. Dentin matrix protein 1 (DMP1) is one such protein whose loss-of-function results in a human disease phenotypically identical to XLH. Using PHEX-deficient dental pulp stem cells (DPSCs) from XLH patients, we have demonstrated impaired processing of ECM proteins and elevated levels of matrix metalloprotease 3 (MMP3). Constitutive expression of DMP1 or the addition of differentiation medium containing phosphate revealed normal protein processing in XLH DPSCs. The following specific aims are designed to investigate the influence of phosphate and DMP1 in restoring the impaired dentin ECM in XLH pathology. Using DPSCs from healthy and XLH patients, we will establish cell lines overexpressing different forms of DMP1. By manipulating in vitro conditions, we will evaluate the effect of phosphates and DMP1 on the genetic and proteomic profile of healthy and XLH DPSCs, focusing on the ECM proteins that constitute the dentin matrix (Aim 1). In parallel, we will generate a DMP1-overexpressing Phex-deficient mouse model by crossing our tooth-specific dentinsialophosphoprotein (Dspp) promoter-driven DMP1 transgenic mouse with the Hyp mouse. The effect of DMP1-overexpression on the spatiotemporal distribution of ECM proteins and the mineral quality of tooth dentin will be evaluated (Aim 2). The proposed studies will address the local effects of PHEX mutations on dentin matrix deposition and will further clarify the predicted relationship between PHEX, DMP1, and phosphate. The studies will also address the possibility of using DMP1-mediated treatments as targeted therapies for XLH. This research will be conducted at a dental college within the University of Illinois at Chicago?s medical district campus, an ideal research environment for the fulfillment of the applicant?s academic and professional goals.